1. Field of the Invention
The present invention relates to a simple and efficient method for the quantitative determination of ligand interactions with receptors adsorbed or immobilized on the surface of a solid material by direct measurement of the reflected light intensity.
More specifically, the present invention refers to a method for the quantitative determination of ligand interactions with receptors wherein planar surfaces of materials, having a refractive index between 1.32 and 1.35, are used.
2. Description of the Related Art
In the prior art, several methods to determine interactions between ligands and receptors, that is the binding affinities of ligand-receptor reversible systems, of chemical, biochemical or biological interest have been reported. A list is reported in Angew. Chem. Int. Ed. 1998, 37, page 2785. The known methods generally comprise the receptor immobilization on a suitable flat surface and the direct or indirect measurement of the variations of certain surface properties, for example the optical ones, after the ligands enter into contact with the surface. The variations are due to the formation of ligand/receptor couples.
One class of these methods requires the labeling of the ligand in solution, that is the covalent modification of the ligand with fluorescent, luminescent or radioactive species (see for example patent application US 2004/0014060 A1). However, it is to be noted that the modification of ligand is very complex and long and difficult to be used in screening tests where numerous different ligands are used. Furthermore, the methods require an additional operation for removing, by washing out, the free ligands, those which have not interacted with the receptors and which interfere with the measurement. A further drawback of said method is that the ligand-receptor interaction can be influenced by the chemical modification of the ligand due to labeling.
Another class of methods which simulated more effectively the ligand-receptor interactions (for example those occurring on a cell membrane surface), directly exploits the variations induced on a surface by the bond formation in the ligand-receptor couple, without modifying the ligand with labeling substances. An example of this method uses the biosensor BIAcore, marketed by GE Healthcare (Uppsala, Sweden). See for example U.S. Pat. No. 5,313,264 and U.S. Pat. No. 5,374,563. In this biosensor, wherein the principle of Surface Plasmon Resonance (SPR) is used (see the publication Jiri Homola, Sinclair S. Yee, Gunter Gauglitz, Surface plasmon resonance sensors: review, Sensors and Actuators B, vol. 54 (1999), pages 3-15), an evanescent optical wave couples with surface plasmons of thin layers (50 nm) of conducting materials such as silver or gold, and generates a resonance phenomenon at specific angles. This allows determining the variation of the refractive index of the layer of immobilized material on the metal, for example a ligand-receptor couple. The binding constants between ligand and receptor are obtained from this variation.
This method, even though it is very used in practice, is rather complicated and expensive and it is not always accurate in the determination of the binding constants. See for example the publication “Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules”, by Peter Schuck, Annu. Rev. Biophys. Biomol. Struct., 1997, 26, pages 541-566. In fact, said method is based on an indirect detection of the adsorbed mass by the effect that it has on the propagation velocity of a plasmon, which in turn determines the coupling angle of a laser beam.
The problems connected to the use of the BIAcore method for the determination of the binding constants mostly depend on the complexity of the method:                the measured signal depends on the physical properties of five different materials through a complex functional dependence including parameters not known a priori; the five mentioned materials are: the glass support or similar products, the thin layer of the conductor deposited on the support, the polymeric layer which allows to functionalize the metallic surface, the molecules adhering because of the interaction and the aqueous solution;        the sensitivity and accuracy of the measurement strongly depend on the thickness and the surface quality of the conductor layer forming the sensor (see the publication “Optical properties and instrumental performances of thin gold films near the surface plasmon resonance” by H. Neff et al., Thin solid films, 2006, 496, pages 688-697);        the measurements are based on the detection of the light intensity at various angles and this requires an equipment capable of high resolution angular scanning and thus it is composed by high precision moving parts or photodetector matrices of suitable space resolution (see the article “Quantitative interpretation of the response of surface plasmon resonance sensors to adsorbed films” by L. S. Jung et al, Langmuir, 1998, 14, pages 5636-5648).Said problems produce:        the non agreement between the affinity constant values determined through the binding kinetics and those obtained at the thermodynamic equilibrium;        the impossibility to predict the intensity of the signal generated when ligand/receptor couples are formed on the surface, since the signal depends on not previously known parameters.        
The need was therefore felt to have available a simple method for the determination of interactions between ligands and receptors directly exploiting the variations induced by the ligand-receptor interaction on a surface, avoiding the ligand labeling operations, allowing the determination of the affinity constant values under thermodynamic equilibrium conditions, thus avoiding the drawbacks of the indirect methods, such as, for example, BIAcore, and allowing the use of the method also in the study of multivalent ligands, since most of the ligands of biological and pharmacological interest have multiple binding sites. In particular, the need was felt for a high sensitivity method, whose signal was detectable by means of instrumentation simple to build, and was quantitatively interpretable through previously known parameters, overcoming, in this way, the prior art drawbacks.